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Detection of dideoxyosone intermediates of glycation using a monoclonal antibody: Characterization of major epitope structures
Archives of Biochemistry and Biophysics 446 (2006) 186–196 www.elsevier.com/locate/yabbi
Detection of dideoxyosone intermediates of glycation using a monoclonal antibody: Characterization of major epitope structures Shivaprakash Puttaiah a, Yuming Zhang b, Heather A. Pilch a, Christoph Pfahler a, Tomoko Oya-Ito a, Lawrence M. Sayre b, Ram H. Nagaraj a,c,¤ a
Department of Ophthalmology, Case Western Reserve University, Cleveland, OH 44106, USA b Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106, USA c Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44106, USA Received 30 September 2005, and in revised form 1 December 2005 Available online 27 December 2005
Abstract Glycation or the Maillard reaction in proteins forms advanced glycation end products (AGEs) that contribute to age- and diabetesassociated changes in tissues. Dideoxyosones, which are formed by the long-range carbonyl shift of the Amadori product, are newly discovered intermediates in the process of AGE formation in proteins. They react with o-phenylenediamine (OPD) to produce quinoxalines. We developed a monoclonal antibody against 2-methylquinoxaline-6-carboxylate coupled to keyhole limpet hemocyanin. The antibody reacted strongly with ribose and fructose (+OPD)-modiWed RNase A and weakly with glucose and ascorbate (+OPD)-modiWed RNase A. Reaction with substituted quinoxalines indicated that this antibody favored the 2-methyl group on the quinoxaline ring. We used high performance liquid chromatography to isolate and purify three antibody-reactive products from a reaction mixture of N-hippuryl1 6 L-lysine + ribose + OPD. The two most reactive products were identiWed as diastereoisomers of N -benzoylglycyl-N -(2-hydroxy-3-quinoxalin-2-ylpropyl)lysine and the other less reactive product as N1-benzoylglycyl-N6-[2-hydroxy-2-(3-methylquinoxalin-2-yl)ethyl]lysine. Our study conWrms that dideoxyosone intermediates form during glycation and oVers a new tool for the study of this important pathway in diabetes and aging. 2005 Elsevier Inc. All rights reserved. Keywords: Glycation; AGEs; Diabetes; Aging; Dideoxyosones; Quinoxalines; Monoclonal antibody
Non-enzymatic glycation, or the Maillard reaction, between amino groups in amino acids and carbonyl compounds (such as, sugars, ascorbate oxidation products, and dicarbonyls) produces a reversible condensation product known as the Amadori product. This product, through a series of reactions, generates irreversible adducts on proteins that are collectively known as advanced glycation end products or AGEs [1–3]. Several AGE structures have been elucidated, and many have been detected in tissues of man and animals [4,5]. AGEs accumulate in various tissues during aging and accumulate to higher degree in diabetes, espe-
*
Corresponding author. Fax: +1 216 844 7962. E-mail address: [email protected] (R.H. Nagaraj).
0003-9861/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2005.12.002
cially in long-lived proteins, such as collagen and lens crystallins [6–9]. The formation of AGEs in proteins has been implicated in a number of age-related diseases, such as cataract [10–12] and Alzheimer’s disease [13] and also in complications of diabetes, such as retinopathy [14] and nephropathy [15]. AGEs accumulate in tissues at a higher rate in individuals with diabetes because of higher blood sugar and increased oxidative stress-associated with that disease. Many studies indicate a positive association between accumulation of AGEs, especially in skin collagen, and the severity of complications in diabetes [16,17]. In fact, recent studies suggest that AGEs in skin could be surrogate markers for both macro- and microvascular complications in diabetes [18]. Additionally, several studies show that
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Fig. 1. A scheme depicting the possible pathways of synthesis AGEs through the dideoxyosone intermediates.
treatment of diabetic animals with putative AGE inhibitors can reduce or prevent the biochemical and morphological changes reminiscent of the disease in humans [19,20]. Despite convincing evidence that AGEs play a role in aging and diabetes, the chemical pathways of AGE formation in vivo are not clear, except for a few AGEs, such as, N-carboxymethyllysine (CML).1 It is now well accepted that dicarbonyl compounds, such as, methylglyoxal and 3deoxyglucosone, are the major intermediates during AGE synthesis. Several years ago, Lederer discovered another novel pathway, in which the Amadori product of glycation spontaneously undergoes a long-range carbonyl shift to produce dideoxyosones such as N6-(2,3-dihydroxy-5,6dioxohexyl)lysine [21] (Fig. 1). Because of the vicinal carbonyl functions, these intermediates can condense with guanidino groups in arginine to crosslink amino acids. A few AGEs derived from this pathway have been identiWed in tissue proteins, including lens proteins [22–24]. This pathway appears to be a major source for AGEs, because protein-bound dideoxyosones are likely to escape enzymatic degradation. Because dideoxyosones are highly reactive transient intermediates of AGE formation, stabilizing derivatization would be necessary for their detection in tissue proteins. Lederer’s group used aminoguanidine and o-phenylenediamine (OPD) to trap dideoxyosone intermediates and concluded that the latter is more eYcacious than the former [25]. When OPD reacts with dideoxyosones, it forms quinoxalines on proteins. We took advantage of this property 1 Abbreviations used: CML, N-carboxymethyllysine; OPD, o-phenylenediamine; sulfo-NHS, N-hydroxysulfosuccinimide; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; KLH, keyhole limpet hemocyanin.
and developed a monoclonal antibody against 2-methylquinoxaline-6-carboxylate chemically coupled to KLH. This paper describes detection of protein-bound quinoxalines with our novel antibody and we report the epitope structures detected by the antibody in proteins modiWed by sugars and OPD. Materials and methods Materials Methylglyoxal, o-phenylenediamine (OPD) BSA, RNase A, N-hippuryl-L-lysine, sugars, and L-ascorbate were from Sigma Chemical, St. Louis, MO. All quinoxalines and 2,3-diaminobenzoic acid were obtained from Aldrich Chemical, Milwaukee, WI. N-Hydroxysulfosuccinimide (sulfo-NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and keyhole limpet hemocyanin (KLH) were from Pierce Biotechnology, Rockford, IL. Synthesis of methylquinoxaline-6-carboxylate (3) O
H2N H
COOH
1
N +
H2N
O
N
+ HO2C
2
N
3a (70%)
HO2C
N
3b (30%)
A solution of 3,4-diaminobenzoic acid (2) (380 mg, 2.5 mmol) in methanol (20 ml) was added to methylglyoxal aqueous solution (1) (580 l, 3.8 mmol). The reaction mixture was stirred at room temperature for 2 h. The solvent was then removed under reduced pressure, and the residue was dissolved in methanol. Silica gel was added, and the
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methanol was removed. Silica gel was poured into a glass column, and the bound material was eluted with ethyl acetate:methanol (9:1, v/v). The eluate was monitored by thin layer chromatography (solvent system ethyl acetate/methanol 9:1). The Rf value for the product was 0.1. Fractions with products were pooled, the solvent evaporated, and the mixture was suspended in methanol, absorbed on silica gel and re-chromatographed as before. Fractions with products were combined, dried and stored at 4 °C until use. The puriWed product was found to be an isomeric mixture of 3a and 3b in a 7:3 ratio (»70% yield). The 1H NMR spectrum showed the following characteristics. 3a (major): 1H NMR (DMSO) 8.96 (s, 1H), 8.58 (d, J D 1.8 Hz, 1H), 8.27 (dd, J D 8.8, 2.0 Hz, 1H), 8.09 (d, J D 8.7 Hz, 1H), 2.73 (s, 3H). 3b (minor): 1H NMR (DMSO) 8.96 (s, 1H), 8.52 (d, J D 1.8 Hz, 1H), 8.24 (dd, J D 8.6, 1.8 Hz, 1H), 8.14 (d, J D 8.8 Hz, 1H), 2.73 (s, 3H). The m/z of the product as the trimethylsilyl (TMS) derivative determined by gas chromatography/mass spectrometry was 260, which is compatible with single TMS derivatization on the carboxyl group of 2-methylquinoxaline-6-carboxylate (3a) and 3-methylquinoxaline-6-carboxylate (3b). Conjugation of (3) to keyhole limpet hemocyanin Conjugation was achieved using carbodiimide. Thirty milligrams of (3) was dissolved in 500 l DMSO, and the sample was resuspended in 1.0 ml of 0.1 M MES buVer (pH 6.0). EDC (28.8 mg) was then added in 350 l of water, followed by 2.2 mg of sulfo-NHS in 50 l of water and mixed well. Finally, 20 mg/ml of keyhole limpet hemocyanin (KLH) was added, and the mixture was incubated at room temperature for 4 h. The protein was then extensively dialyzed against PBS. A similar conjugation was made with RNase A to give RNase A-(3). Preparation of monoclonal antibody Mice were injected intraperitoneally with 30 g KLH-(3) in Freund’s complete adjuvant; the initial sensitization was followed with three booster injections of 30 g antigen in incomplete adjuvant at 3 weeks intervals. Serum antibody titers were checked by a direct ELISA. For this determination, 96-well microplate wells were coated with RNase A-(3), and diluted antiserum was incubated with HRP-conjugated rabbit anti-mouse IgG. The mouse with the highest antibody titer was chosen for hybridoma production. Mouse spleen cells were fused with P3U1 myeloma cells by a standard method [26]. Hybridoma supernatants were screened by ELISAs using two diVerent coating solutions. The microplate wells were coated with either 1 g/well RNase A-(3) in 0.05 M sodium carbonate buVer (pH 9.7) (BuVer A) or with 1 g/well RNase A + ribose + OPD in BuVer A. RNase A + ribose + OPD was prepared by incubating 10 mg/ml RNase A + 200 mM ribose + 50 mM OPD + 2 mM EDTA + 2 mM DTPA in 0.1 M phosphate buVer (pH 7.4) at 37 °C for 7 days, followed by dialysis
against PBS for 48 h. After blocking with 5% non-fat dry milk (NFDM), 50 l of hybridoma supernatant was added to wells and incubated for 1.5 h, and then washed 3 times with PBS + 0.05% Tween 20 (PBST). The wells were then incubated for 2 h with 50 l of HRP-conjugated rabbit anti mouse IgG (1:5000 dilution). The wells were washed 3 times with PBST and incubated with 3,3⬘,5,5⬘-tetramethylbenzidine substrate at 37 °C. The reaction was stopped by addition of 50 l of 2 N H2SO4, and the color of the product at 450 nm was measured spectrophotometrically. Hybridomas with the strongest reaction against both RNase A-(3) and (RNase A + ribose + OPD) samples were subcloned by serial dilution until all the subclones were immunoreactive. The medium was collected from large-scale cultures of hybridoma-producing subclones, and the antibody was puriWed on a Protein G–Sepharose column (Amersham Biosciences, Piscataway, NJ). The antibody was identiWed and classiWed as IgG1. Incubation of proteins with carbohydrates and OPD Solutions of BSA or RNase A (10 mg/ml) in 0.1 M sodium phosphate buVer (pH 7.4) were Wltered through a 0.2 m Wlter. Glucose or ribose was added to the protein solution to obtain a Wnal concentration of 500 mM, and the samples were incubated at 45 °C overnight. The following day, OPD (100 mM stock solution prepared by dissolving 10.8 mg in 1.0 ml of DMSO) was added to a Wnal concentration of 100 mM, and the samples were incubated for an additional 4 h at 45 °C. The proteins were dialyzed against PBS for 24 h with one change of buVer after 16 h. They were then stored at 4 °C until use. In another experiment, RNase A was incubated with several carbohydrates in the presence or absence of OPD. A stock solution of RNase A was prepared by dissolving 100 mg in 20 ml of 0.1 M phosphate buVer (pH 7.4). This solution was then Wltered through a 0.2 m Wlter, and 1.0 ml was added to each sample. Duplicate samples included unaltered RNase A controls and samples containing 500 mM of D-glucose, D-fructose, D-ribose, or L-ascorbic acid. All the samples were incubated at 45 °C for 16 h. One set of samples was treated with 100 M OPD stock solution (1.0 mg/1.0 ml in 0.1 M phosphate buVer, pH 7.4) and incubated at 35 °C for 16 h. Sodium borohydride reduction of ribose-modiWed RNase A RNase A 10 mg/ml in 0.1 M sodium phosphate buVer pH 7.4 was Wltered through a 0.2 Wlter. Five hundred millimolar ribose was then added and incubated at 37 °C for 24 h. The unbound sugar was removed by Wltering through a 10 kDa cut oV centrifugal Wlter (Millipore corporation, Bedford, MA). The centrifugation (4000g for 10 min) was repeated three times, each time adding 0.5 ml of incubation buVer to the sample. The pH of the sample was adjusted to 8 using 2 N NaOH. The sample was then divided into two equal portions, to one, 100 mM sodium
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borohydride (NaBH4) was added and both portions were incubated at room temperature for 10 min, followed by incubation on ice for 1 h. One hundred millimolar OPD (dissolved in 25 l DMSO) was then added to each, and incubated for an additional 16 h and dialyzed against PBS for 48 h with change of the buVer after 24 h. Protein concentration was estimated by the Bradford Assay. Samples were tested for reaction with the antibody by a direct ELISA (see below). EVect of N-acetyl-L-arginine RNase A (10 mg/ml) in 0.1 M sodium phosphate buVer, pH 7.4, was Wltered through a 0.2 m Wlter. Five hundred millimolar ribose was then added to the solution and incubated for 24 h at 37 °C. The sample was divided into two parts, to one part 100 mM N-acetyl-L-arginine was added. The other part served as control. The samples were further incubated at 37 °C for 16 h. One hundred millimolar OPD (dissolved in 25 l DMSO) was then added to each and incubated for further 16 h at 37 °C. The samples were dialyzed against PBS for 48 h with change of buVer after 24 h and tested for reaction with the antibody by a direct ELISA (see below). Direct enzyme-linked immunosorbent assay (ELISA) Microplate wells were coated with proteins in 0.05 M carbonate buVer (pH 9.7) at a concentration of 1 g/well, incubated at 4 °C overnight and then washed three times with PBST. Before use in the assay, all wells were blocked for 2 h at room temperature with 300 l of 5% NFDM in PBS and washed three times with PBST. The monoclonal antibody (1:500 diluted in 0.1% BSA/PBS) was applied (50 l/well) to each well and incubated for 1.5 h at 37 °C. The wells were then washed three times with PBST and incubated with 50 l of horseradish peroxidase-conjugated rabbit anti-mouse IgG (Promega, Madison, WI, diluted 1:2500) for 1.5 h at 37 °C. The enzyme reaction was assessed by addition of 100 l of 3,3⬘,5⬘,5⬘-tetramethylbenzidine (Sigma) followed by addition of 50 l of 2 N H2SO4. Chromophore absorbance was measured at 450 nm. Competitive ELISA All procedures were similar to the direct ELISA except that the test samples were incubated with the monoclonal antibody (1:500 in 0.1% BSA/PBS as Wnal dilution) for 1.5 h at 37 °C before addition (50 l) to the wells. Western blotting RNase A samples incubated as described above were subjected to Western blotting (10 g of protein on 12% reducing gels), and the proteins were electrophoretically transferred to a nitrocellulose membrane 0.45 m (Bio-Rad). Identical gels
189
were stained with BioSafe Coomassie blue (Bio-Rad). The membrane was then blocked with 5% NFDM in PBS for 2 h and incubated at 4 °C for 16 h with shaking. The membranes were washed Wve times with PBST and the monoclonal antibody was applied at a dilution of 1:80 in 5% NFDM in PBST. The samples were incubated with shaking for 3 h at room temperature. The membrane was washed again and incubated for 1.5 h longer with rabbit anti-mouse IgG (dilution 1:7500, Promega, Madison, WI). After washing three times, Supersignal West Pico chemiluminescent substrate (Pierce) was applied to the membranes for 10 min, then they were exposed to X-ray Wlm (Pierce, CL-XPosure Film). Isolation of antibody-reactive products from reaction mixture of N-hippuryl-L-lysine + ribose + OPD N-hippuryl-L-lysine (6 mmol) and DTPA (0.013 mmol) were dissolved completely in 2.5 ml of 0.1 M sodium phosphate buVer (pH 7.4). OPD (4 mmol) was dissolved separately in 100 l of DMSO and added to the incubation mixture. D-ribose (2 mmol) was then added to the mixture, and the volume was adjusted to 5.0 ml by addition of 2.5 ml of 0.1 M phosphate buVer (pH 7.4). The mixture was incubated at 70 °C for 48 h. A sample (60 l) of reaction mixture was injected into a C18 semi-preparative reversed phase column (Vydac, 218TP1010). A linear gradient was established with solvent A (0.1% triXuoroacetic acid in water) and solvent B (50% acetonitrile and 0.1% triXuoroacetic acid in water). The following program was applied: 0–5 min, 0% B; 5–60 min, 0–100% B; 60–67 min, 100% B; 68–75 min, 0% B at a Xow rate of 2.0 ml/min. We used an online UV detector (Jasco, Tokyo, Japan, Model UV-970) to monitor the column eZuent for absorbance at 228 nm. Absorbance from quinoxaline-like products was also monitored at 325 nm. Based on the chromatograms, we identiWed six peaks (peaks 2–7) as possible quinoxalines (Fig. 7). They were collected and dried in a Savant Speedvac concentrator (Savant Instruments, Hicksville, NY), re-suspended in 250 l of water and tested by competitive ELISA (only 2–6 were tested, 7 was in insuYcient quantity). 1 H NMR spectroscopy was performed on either Varian Gemini 200 or Varian Inova 400 Instruments (with chemical shifts referenced to TMS or the solvent peak). High-resolution mass spectra (HRMS) were obtained at 20–40 eV on a Kratos MS-25A instrument. Results We planned to detect dideoxyosones in glycated proteins by trapping them with OPD (Fig. 2). This reaction produces quinoxalines. e.g., with glucose derived dideoxyosone, the OPD reaction produces N6-(2,3-dihydroxy-4-quinoxalin-2-ylbutyl)lysine. However, if the Amadori is derived from ribose, the resulting dideoxyosone, upon reaction with OPD would produce N6-(2-hydroxy-3-quinoxalin-2-ylpropyl)lysine and possibly N6-[2-hydroxy-2-(3-methylquinoxalin-2-yl)ethyl]lysine [22,23]. We wanted to raise an antibody
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Fig. 2. Trapping of dideoxyosone intermediates by OPD. Structures in the box were ones we wanted to detect.
that recognized only the quinoxaline moiety, irrespective of substitutions on the quinoxaline core. Accordingly, we synthesized 2-methylquinoxaline-6-carboxylate for conjugation with KLH. The KLH-quinoxaline was then used as an antigen to stimulate production of the monoclonal antibody. To develop an ELISA for screening proteins, we Wrst tested immunoreactivity of BSA and RNase A following incubation with glucose or ribose and OPD. Fig. 3A shows the results of a direct ELISA with 1 g of the protein/well. RNase A + ribose + OPD showed the highest immunoreactivity followed by BSA + ribose + OPD. Proteins modiWed by glucose and OPD were relatively poor reactants. To test if the antibody reacted non-speciWcally with ribose modiWed RNase A, we performed a direct ELISA. The idea was to reduce DDOs with NaBH4 and prevent their reaction with OPD to form quinoxalines. The ELISA procedure involved coating of microplate wells with 1 g of NaBH4 reduced ribose-modiWed RNase A (and treated with OPD) or nonreduced ribose-modiWed RNase A (treated with OPD). The results are shown in Fig. 3B. Antibody failed to react with the reduced protein, but reacted with non-reduced protein. The data suggest that the antibody requires quinoxalines for reaction in glycated proteins. Since the antibody reacted stronger with RNaseA + ribose + OPD (Fig. 3A), we used this preparation in all our subsequent ELISAs. To test the formation of dideoxyosones with various sugars and ascorbate oxidation products, we incubated RNase
Fig. 3. (A) Reactivity of RNase A modiWed by glucose or ribose (+OPD). RNase A was incubated with sugars in the presence of OPD and tested for reaction with antibody by a direct ELISA. (B) Reactivity of NaBH4 reduced RNase A + ribose + OPD. RNase A was incubated with ribose and reduced with NaBH4 before addition of OPD.
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A with glucose, fructose or ascorbate in the presence or absence of OPD. Fig. 4A shows that among these three carbohydrates, fructose produces the greatest quantity of dideoxyosones, followed by glucose and ascorbate. Samples with fructose + OPD (10 g protein/well) inhibited antibody binding by nearly 80%. At this concentration of protein, binding was inhibited 72% by glucose + OPD and 52% by ascorbate + OPD. Samples containing no OPD, RNase A + OPD, or RNase A alone reacted only weakly with the antibody. The mild reaction of the antibody with glycated proteins (without OPD) suggested the possibility of non-speciWc reaction, possibly with AGEs. We tested this possibility by performing a direct ELISA using RNase A + ribose + Nacetyl-L-arginine sample. This sample is expected to contain signiWcant quantities of AGEs, including DDO-derived lysine–arginine crosslinking structures. Our antibody failed to react with this preparation (Fig. 4B). These data suggest that the mild immunoreactivity of glycated proteins (without OPD) may not be due to arginine-derived AGEs. Formation of ‘quinoxaline-like’ products without the involvement of arginine is a possibility. However such products, even if formed, may not signiWcantly interfere with detection of DDO-derived quinoxalines as the reaction with glycated pro-
191
teins (without OPD) was 80–90% lower than reaction with glycated proteins (with OPD) (Fig. 4A). We used Western blotting to conWrm the ELISA results. As shown in Fig. 5, all proteins that were modiWed by carbohydrates and OPD showed immunoreactivity. The non-crosslinked RNase A monomer accounted for most of the immunoreactivity, with some in the dimeric protein. These results conWrm our Wndings with ELISA and suggest that OPD curtails crosslinking of proteins through dideoxyosone intermediates. Furthermore, these results indicate that dideoxyosones form not only from sugars, but also from ascorbate, possibly from ascorbate oxidation products. We characterized the antibody further using commercial quinoxalines (0–2000 pmol) in a competitive ELISA. Fig. 6 shows that most quinoxalines reacted with the antibody; the exception was quinoxaline-2-carboxylate. Reaction with 2-methylquinoxaline was strongest, with 100% inhibition of the antibody binding to wells at 500 pmol. Quinoxaline and 5-methylquinoxaline were less eVective than 2-methylquinoxaline and required four times as much (2000 pmol/well) to achieve 100% inhibition of the antibody. Other quinoxalines such as 2-quinoxalinol, 2,3-
Fig. 4. (A) Immunoreactivity of RNase A modiWed by sugars and ascorbate in the presence or absence of OPD. RNase A was incubated Wrst with 500 mM concentrations of sugars or ascorbate for 12 h and then with 1.0 mM OPD at 37 °C and pH 7.4 for 16 h. Proteins were tested in a competitive ELISA using 0, 2, and 4 g protein. (B) EVect of addition of N-acetyl-L-arginine on the immunoreactivity of glycated protein. RNase A was incubated with ribose for 24 h, 100 mM OPD or N-acetyl-L-arginine was then added to the reaction mixture and incubated for 16 h. The reaction mixtures were dialyzed against PBS and tested by direct ELISA as described in Materials and methods.
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Fig. 5. Western blotting of RNase A modiWed by sugars or ascorbate (§OPD). Proteins were incubated under conditions described in Fig. 4. Ten micrograms protein was subjected to SDS–PAGE on a 12% reducing gel and transferred to a nitrocellulose membrane. The membrane was incubated Wrst with the monoclonal antibody followed by incubation with HRP-conjugated rabbit anti-mouse IgG and developed by the Pierce Enhanced Chemiluminescence kit. (A) is a Coomassie stained gel, and (B) is a Western blot of the same material. Lane 1, molecular weight markers; lane 2, RNase A; lane 3, RNase A + OPD; lane 4, RNase A + glucose; lane 5, RNase A + glucose + OPD; lane 6, RNase A + ribose; lane 7, RNase A + ribose + OPD; lane 8, RNase A + fructose; lane 9, RNase A + fructose + OPD; lane 10, RNase A + ascorbate; lane 11, RNase A + ascorbate + OPD.
Fig. 6. Immunoreactivity of quinoxalines with the antibody. Commercially available quinoxalines (0–2000 pmol) were tested in competitive ELISA.
dimethylquinoxaline, and quinoline were comparable, producing 80–90% inhibition at 2000 pmol/well. From these results, we concluded that the antibody favors 2-methyl substitution on quinoxaline for maximum reactivity (Fig. 6, inset).
To determine the possible epitope structures in lysine-derived dideoxyosones, we attempted to isolate antibody-reactive structures following the incubation of Nhippuryl-L-lysine with ribose and OPD. The reaction mixture was subjected to HPLC on a C18 reversed phase column. The column eluate was monitored for absorbance at 228 nm (for the hippuryl group) or 325 nm (for quinoxalines). Several peaks were observed as shown in Fig. 7. Peaks 2–7 were observed at both 228 and 325 nm, indicating that they could be quinoxalines. These peaks were collected from 2 injections and dried in a Savant Speed Vac Concentrator. The dried fractions were re-suspended in 0.25 ml of dH2O. From each fraction, 100 l of sample was mixed with 100 l of 6 N HCl and incubated at 110 °C for 16 h. After acid hydrolysis, drying of samples and reconstitution in 100 l of dH2O, 20 l from each was spotted on a silica gel thin layer chromatography plate. The unhydrolyzed portion of the fractions was spotted on a separate plate and developed by spraying ninhydrin reagent (2% in ethyl alcohol) onto each plate. Fractions 4, 5, and 6 were weakly ninhydrin positive after acid hydrolysis, indicating that these peaks contain lysine. Products in peaks 2 and 3 were ninhydrin negative and thus are probably lysine-free quinoxalines, formed from the reaction of deoxyosone intermediates of glycation with OPD. We tested products in peaks 4–6 in a competitive ELISA to determine if they reacted with the antibody. The results show that the product in peak 5 was most reactive with nearly 75% inhibition of antibody binding at 0.4 l (Fig. 8). Products in peak 4 and 6 also inhibited antibody binding, but were less eVective than the product in peak 5. We acknowledge that these measurements are not quantitative, because product concentrations presumably diVer within the peaks. Based on these results, we collected products in peaks 4, 5, and 6 (henceforth referred to as compounds 4, 5, and 6) from 30 injections of 60 l each. These samples were concentrated by lyophilization and re-injected into the HPLC column. The separated compounds were then collected, freeze-dried and dissolved in 0.5 ml methanol. The 1H NMR spectrum for 4 and 5 showed the following signals: 4: 1H NMR (CD3OD) 9.07 (s, H-13), 8.15– 8.05 (2H, H-14,17), 7.90–7.80 (4H, H-3,15,16), 7.55–7.40 (3H, H-1,2), 4.50–4.30 (2H, H-5,11), 4.10–4.00 (m, 2H, H-
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Fig. 7. Isolation and puriWcation of immunoreactive products from reaction mixture of N-hippuryl-L-lysine + ribose + OPD by HPLC. HPLC was performed on a C18 reversed phase semi-preparative column using a water/acetonitrile (with 0.1% TFA) gradient program. The column eluate was monitored for absorbance at 228 nm (for the hippuryl group, A) and 325 nm (for quinoxalines, B). Peaks 4–7 were collected by repeated injections. The products in these peaks were further puriWed on the same column as described in Materials and methods.
Fig. 8. Immunoreactivity of HPLC puriWed products. The products puriWed from HPLC (peaks 4–6) were freeze-dried and dissolved in DMSO. Samples at indicated volumes were tested for reaction with the antibody in a competitive ELISA.
4), 3.20–2.90 (6H, H-9,10,12), 2.00–1.40 (6H, H-6,7,8); 5: 1 H NMR (CD3OD) 8.80 (s, H-13), 8.20–8.00 (2H, H14,17), 7.90–7.70 (4H, H-3,15,16), 7.60–7.40 (m, 3H, H-1,2),
4.60–4.45 (2H, H-5,11), 4.10–3.90 (m, 2H, H-4), 3.20–2.90 (6H, H-9,10,12), 2.00–1.40 (6H, H-6,7,8). Fig. 9A shows the 1H NMR spectrum for 5, the one for 4 is nearly identical (not shown). Compounds 4 and 5 are diastereoisomers caused by two chiral centers, C-5 and C-11. The proton signals of 4 and 5 around 9.00 ppm were assigned to H-13 due to the strong deshielding eVect of the quinoxaline ring. The resonances around 7.5–8.2 ppm region are due to the hippuryl and quinoxaline moieties. The high Weld aliphatic hydrogen signals between 2.00 and 1.40 ppm are attributed to the lysine side chain internal methylenes. HRMS (FAB) for 5 showed a m/z of 494.2390, which was similar to the calculated m/z for C26H32N5O5 (MH+) of 494.2403. The UV spectrum of 5 showed max of 325 nm, which was attributable to the quinoxaline ring (Fig. 9B). The UV spectrum for 4 was identical to that of 5. Based on these characteristics, the product was identiWed as, N1benzoylglycyl-N6-(2-hydroxy-3-quinoxalin-2-ylpropyl)lysine (structure shown in Fig. 9A inset). The 1H NMR analysis of 6 showed the following signals (Fig. 10A): 1H NMR (CD3OD) 8.20–7.90 (2H, H14,17), 7.90–7.70 (4H, H-3,15,16), 7.60–7.30 (3H, H-1,2), 5.40–5.30 (m, 1H, H-11), 4.70–4.50 (m, 1H, H-5), 4.20–3.90
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Fig. 9. 1H NMR spectrum of 5. Compound 4 displayed a nearly identical spectrum to 5. Based on the 1H NMR characteristics, the structure of the product is shown in the inset. UV spectrum is shown in the (B).
(m, 2H, H-4), 3.80–3.10 (4H, H-9,10), 2.90 (s, 3H, H-12), 2.10–1.50 (6H, H-6,7,8). The structure of 6 was determined by the speciWc positions of H-11 and H-12 besides its aromatic protons attributed to the hippuryl and quinoxaline groups. Due to the deshielding eVect of quinoxaline ring and electronegativity of hydroxyl group, H-11 resonates downWeld around 5.3 ppm. The 12-CH3 singlet appears at 2.9 ppm, characteristic of the methyl group at position 2 on the quinoxaline ring. HRMS (FAB) showed a m/z of 494.2406, which was fully agreeable with the calculated m/ z for C26H32N5O5 (MH+) of 494.2403. The UV spectrum of 6 showed max of 325 nm, which was due to the quinoxaline ring (Fig. 10B). Based on these characteristics, we identiWed the product as N1-benzoylglycyl-N6-[2-hydroxy2-(3-methylquinoxalin-2-yl)ethyl]lysine (structure shown in Fig. 10A inset). Fig. 11 indicates the potential mechanism of formation of quinoxalines from the reaction of N-hippuryl-L-lysine + ribose + OPD. Discussion Dideoxyosones are likely to be major intermediates during AGE synthesis in vivo, yet little is known about the chemical pathways of AGE formation from these intermediates. Only two dideoxyosone-derived AGEs, pentosidine
and glucosepane, are hitherto known; they are both crosslinking structures. Biemel and Lederer [25] suggested that glucosepane accounted for only a fraction of the total dideoxyosone formed from the reaction of glucose, and they speculated that dideoxyosone could generate a variety of other protein crosslinks. In this regard, detection of dideoxyosones is an important step towards understanding how this pathway contributes to the pathogenesis of diabetic and age-associated complications. Biemel and Lederer [25] showed that OPD was better than aminoguanidine in trapping dideoxyosones. Accordingly, we chose OPD to trap dideoxyosones. We reasoned that some quinoxalines might have 2-methyl group substitution as shown in Fig. 2. We wanted to detect dideoxyosones (as quinoxalines) formed from not just sugars, but also from ascorbate oxidation products. Taking these factors into consideration, we used 2-methylquinoxaline-6carboxylate as an antigen. The antigen was coupled to KLH through the COOH group. We found that this monoclonal antibody recognized a variety of quinoxalines, but the favored structure was 2-methylquinoxaline. We assumed that when dideoxyosones formed on proteins react with OPD, they would generate quinoxalines with the sugar moiety attached to the pyrazine ring. However, our antigen, 2-methylquinoxaline-6-carboxylate was
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195
Fig. 10. 1H NMR spectrum of 6. UV spectrum is shown in (B).
coupled to protein through the carboxyl group on the benzene rather than pyrazine ring. This diVerence may preclude or weaken antigen detection in biological specimens. However, the fact that we could detect quinoxalines in proteins incubated with sugars (+OPD) suggests that the antibody recognizes ‘biological’ quinoxalines, and it might prove useful for assessing the impact of dideoxyosones in AGE formation in vivo. Formation of greater quantities of dideoxyosones from fructose than from glucose may reXect the higher reactivity of fructose with lysine. Fructose-mediated glycation is reported to produce at least 10 times greater crosslinking than glucose-mediated glycation [27]. The formation of dideoxyosones from ascorbate is not totally unexpected, because ascorbate through oxidation forms sugars, such as threose [28], which can potentially form dideoxyosones, like ribose and glucose. Our Western blot experiment showed that the antibody recognizes primarily the modiWed monomer of RNase A. Coomassie staining conWrmed that a larger portion of the protein remained as a monomer after reaction with sugars and OPD relative to reaction of sugars in the absence of OPD. This suggests that signiWcant pro-
tein crosslinking occurs through dideoxyosone intermediate formation and that OPD inhibits protein crosslinking by binding to dideoxyosones. The identiWcation of N1-benzoylglycyl-N6-(2-hydroxy-3quinoxalin-2-ylpropyl)lysine and N1-benzoylglycyl-N6-[2hydroxy-3-(3-methylquinoxalin-2-yl)ethyl]lysine as major antigens from the reaction of N-hippuryl lysine + ribose + OPD conWrms that the precursor dideoxyosone lysine adducts, capable of crosslinking to protein arginine residues, form from ribose during glycation. Whether these are the major products generated during AGE formation in vivo remains to be established. We believe that quinoxaline-antibody detection of dideoxyosone intermediates oVers considerable advantages over conventional chromatographic methods. The antibody can recognize quinoxalines of dideoxyosones derived from a variety of sugars, and it can be used to quantify dideoxyosones derived from various Maillard reaction pathways. The ability of our monoclonal antibody to detect dideoxyosone intermediates as quinoxaline derivatives oVers a rapid screening method for future research as well as the potential for therapeutic agents that could be used against AGE
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Fig. 11. Possible pathways for the formation of quinoxalines (4, 5, and 6) from the reaction of N-hippuryl-L-lysine, ribose, and OPD.
formation from dideoxyosones intermediates. Such agents would be an important addition to the therapeutic armamentarium against diabetes. Acknowledgments This study was supported by NIH Grants R21DK068045, R01EY-09912, and R01EY-016219 (R.H.N.), P30EY-11373 (Visual Sciences Research Center of CWRU), R01-AG14249 (L.M.S.), Research to Prevent Blindness, NY and Ohio Lions Eye Research Foundation. References [1] S.R. Thorpe, J.W. Baynes, Amino Acids 25 (2003) 275–281. [2] M. Brownlee, Metabolism 49 (2000) 9–13. [3] A.W. Stitt, A.J. Jenkins, M.E. Cooper, Expert Opin. Invest. Drugs 11 (2002) 1205–1223. [4] M.C. Wells-Knecht, S.R. Thorpe, J.W. Baynes, Biochemistry 34 (1995) 15134–15141. [5] H. Vlassara, M.R. Palace, J. Intern. Med. 251 (2002) 87–101. [6] E.B. Frye, T.P. Degenhardt, S.R. Thorpe, J.W. Baynes, J. Biol. Chem. 273 (1998) 18714–18719. [7] J.A. Dunn, D.R. McCance, S.R. Thorpe, T.J. Lyons, J.W. Baynes, Biochemistry 30 (1991) 1205–1210. [8] D.G. Dyer, J.A. Dunn, S.R. Thorpe, K.E. Bailie, T.J. Lyons, D.R. McCance, J.W. Baynes, J. Clin. Invest. 91 (1993) 2463–2469. [9] F.A. Shamsi, A. Partal, C. Sady, M.A. Glomb, R.H. Nagaraj, J. Biol. Chem. 273 (1998) 6928–6936. [10] R.H. Nagaraj, D.R. Sell, M. Prabhakaram, B.J. Ortwerth, V.M. Monnier, Proc. Natl. Acad. Sci. USA 88 (1991) 10257–10261. [11] P. Chellan, R.H. Nagaraj, J. Biol. Chem. 276 (2001) 3895–3903. [12] O.K. Argirov, B. Lin, B.J. Ortwerth, J. Biol. Chem. 279 (2004) 6487–6495.
[13] S. Du Yan, H. Zhu, J. Fu, S.F. Yan, A. Roher, W.W. Tourtellotte, T. Rajavashisth, X. Chen, G.C. Godman, D. Stern, A.M. Schmidt, Proc. Natl. Acad. Sci. USA 94 (1997) 5296–5301. [14] A.W. Stitt, Y.M. Li, T.A. Gardiner, R. Bucala, D.B. Archer, H. Vlassara, Am. J. Pathol. 150 (1997) 523–531. [15] Y. Yamamoto, I. Kato, T. Doi, H. Yonekura, S. Ohashi, M. Takeuchi, T. Watanabe, S. Yamagishi, S. Sakurai, S. Takasawa, H. Okamoto, H. Yamamoto, J. Clin. Invest. 108 (2001) 261–268. [16] D.R. Sell, A. Lapolla, P. Odetti, J. Fogarty, V.M. Monnier, Diabetes 41 (1992) 1286–1292. [17] K.J. Wells-Knecht, E. Brinkmann, M.C. Wells-Knecht, J.E. LitchWeld, M.U. Ahmed, S. Reddy, D.V. Zyzak, S.R. Thorpe, J.W. Baynes, Nephrol. Dial. Transplant. 11 (Suppl 5) (1996) 41–47. [18] V.M. Monnier, O. Bautista, D. Kenny, D.R. Sell, J. Fogarty, W. Dahms, P.A. Cleary, J. Lachin, S. Genuth, Diabetes 48 (1999) 870–880. [19] A. Stitt, T.A. Gardiner, N.L. Alderson, P. Canning, N. Frizzell, N. DuVy, C. Boyle, A.S. Januszewski, M. Chachich, J.W. Baynes, S.R. Thorpe, Diabetes 51 (2002) 2826–2832. [20] D.J. Kelly, R.E. Gilbert, A.J. Cox, T. Soulis, G. Jerums, M.E. Cooper, J. Am. Soc. Nephrol. 12 (2001) 2098–2107. [21] K.M. Biemel, J. Conrad, M.O. Lederer, Angew. Chem. Int. Ed 41 (2002) 801–804. [22] K.M. Biemel, D.A. Friedl, M.O. Lederer, J. Biol. Chem. 277 (2002) 24907–24915. [23] K.M. Biemel, O. Reihl, J. Conrad, M.O. Lederer, J. Biol. Chem. 276 (2001) 23405–23412. [24] D.R. Sell, K.M. Biemel, O. Reihl, M.O. Lederer, C.M. Strauch, V.M. Monnier, J. Biol. Chem. 280 (2005) 12310–12315. [25] K.M. Biemel, M.O. Lederer, Bioconjug. Chem. 14 (2003) 619–628. [26] E. Harlow, D. Lane, Antibodies, A Laboratory Manual, Cold Spring harbor Laboratories, New York, 1988. [27] J.D. McPherson, B.H. Shilton, D.J. Walton, Biochemistry 27 (1988) 1901–1907. [28] J.A. Dunn, M.U. Ahmed, M.H. Murtiashaw, J.M. Richardson, M.D. Walla, S.R. Thorpe, J.W. Baynes, Biochemistry 29 (1990) 10964–10970.
Detection of dideoxyosone intermediates of glycation using a monoclonal antibody: Characterization of major epitope structures Shivaprakash Puttaiah a, Yuming Zhang b, Heather A. Pilch a, Christoph Pfahler a, Tomoko Oya-Ito a, Lawrence M. Sayre b, Ram H. Nagaraj a,c,¤ a
Department of Ophthalmology, Case Western Reserve University, Cleveland, OH 44106, USA b Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106, USA c Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44106, USA Received 30 September 2005, and in revised form 1 December 2005 Available online 27 December 2005
Abstract Glycation or the Maillard reaction in proteins forms advanced glycation end products (AGEs) that contribute to age- and diabetesassociated changes in tissues. Dideoxyosones, which are formed by the long-range carbonyl shift of the Amadori product, are newly discovered intermediates in the process of AGE formation in proteins. They react with o-phenylenediamine (OPD) to produce quinoxalines. We developed a monoclonal antibody against 2-methylquinoxaline-6-carboxylate coupled to keyhole limpet hemocyanin. The antibody reacted strongly with ribose and fructose (+OPD)-modiWed RNase A and weakly with glucose and ascorbate (+OPD)-modiWed RNase A. Reaction with substituted quinoxalines indicated that this antibody favored the 2-methyl group on the quinoxaline ring. We used high performance liquid chromatography to isolate and purify three antibody-reactive products from a reaction mixture of N-hippuryl1 6 L-lysine + ribose + OPD. The two most reactive products were identiWed as diastereoisomers of N -benzoylglycyl-N -(2-hydroxy-3-quinoxalin-2-ylpropyl)lysine and the other less reactive product as N1-benzoylglycyl-N6-[2-hydroxy-2-(3-methylquinoxalin-2-yl)ethyl]lysine. Our study conWrms that dideoxyosone intermediates form during glycation and oVers a new tool for the study of this important pathway in diabetes and aging. 2005 Elsevier Inc. All rights reserved. Keywords: Glycation; AGEs; Diabetes; Aging; Dideoxyosones; Quinoxalines; Monoclonal antibody
Non-enzymatic glycation, or the Maillard reaction, between amino groups in amino acids and carbonyl compounds (such as, sugars, ascorbate oxidation products, and dicarbonyls) produces a reversible condensation product known as the Amadori product. This product, through a series of reactions, generates irreversible adducts on proteins that are collectively known as advanced glycation end products or AGEs [1–3]. Several AGE structures have been elucidated, and many have been detected in tissues of man and animals [4,5]. AGEs accumulate in various tissues during aging and accumulate to higher degree in diabetes, espe-
*
Corresponding author. Fax: +1 216 844 7962. E-mail address: [email protected] (R.H. Nagaraj).
0003-9861/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2005.12.002
cially in long-lived proteins, such as collagen and lens crystallins [6–9]. The formation of AGEs in proteins has been implicated in a number of age-related diseases, such as cataract [10–12] and Alzheimer’s disease [13] and also in complications of diabetes, such as retinopathy [14] and nephropathy [15]. AGEs accumulate in tissues at a higher rate in individuals with diabetes because of higher blood sugar and increased oxidative stress-associated with that disease. Many studies indicate a positive association between accumulation of AGEs, especially in skin collagen, and the severity of complications in diabetes [16,17]. In fact, recent studies suggest that AGEs in skin could be surrogate markers for both macro- and microvascular complications in diabetes [18]. Additionally, several studies show that
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Fig. 1. A scheme depicting the possible pathways of synthesis AGEs through the dideoxyosone intermediates.
treatment of diabetic animals with putative AGE inhibitors can reduce or prevent the biochemical and morphological changes reminiscent of the disease in humans [19,20]. Despite convincing evidence that AGEs play a role in aging and diabetes, the chemical pathways of AGE formation in vivo are not clear, except for a few AGEs, such as, N-carboxymethyllysine (CML).1 It is now well accepted that dicarbonyl compounds, such as, methylglyoxal and 3deoxyglucosone, are the major intermediates during AGE synthesis. Several years ago, Lederer discovered another novel pathway, in which the Amadori product of glycation spontaneously undergoes a long-range carbonyl shift to produce dideoxyosones such as N6-(2,3-dihydroxy-5,6dioxohexyl)lysine [21] (Fig. 1). Because of the vicinal carbonyl functions, these intermediates can condense with guanidino groups in arginine to crosslink amino acids. A few AGEs derived from this pathway have been identiWed in tissue proteins, including lens proteins [22–24]. This pathway appears to be a major source for AGEs, because protein-bound dideoxyosones are likely to escape enzymatic degradation. Because dideoxyosones are highly reactive transient intermediates of AGE formation, stabilizing derivatization would be necessary for their detection in tissue proteins. Lederer’s group used aminoguanidine and o-phenylenediamine (OPD) to trap dideoxyosone intermediates and concluded that the latter is more eYcacious than the former [25]. When OPD reacts with dideoxyosones, it forms quinoxalines on proteins. We took advantage of this property 1 Abbreviations used: CML, N-carboxymethyllysine; OPD, o-phenylenediamine; sulfo-NHS, N-hydroxysulfosuccinimide; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; KLH, keyhole limpet hemocyanin.
and developed a monoclonal antibody against 2-methylquinoxaline-6-carboxylate chemically coupled to KLH. This paper describes detection of protein-bound quinoxalines with our novel antibody and we report the epitope structures detected by the antibody in proteins modiWed by sugars and OPD. Materials and methods Materials Methylglyoxal, o-phenylenediamine (OPD) BSA, RNase A, N-hippuryl-L-lysine, sugars, and L-ascorbate were from Sigma Chemical, St. Louis, MO. All quinoxalines and 2,3-diaminobenzoic acid were obtained from Aldrich Chemical, Milwaukee, WI. N-Hydroxysulfosuccinimide (sulfo-NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and keyhole limpet hemocyanin (KLH) were from Pierce Biotechnology, Rockford, IL. Synthesis of methylquinoxaline-6-carboxylate (3) O
H2N H
COOH
1
N +
H2N
O
N
+ HO2C
2
N
3a (70%)
HO2C
N
3b (30%)
A solution of 3,4-diaminobenzoic acid (2) (380 mg, 2.5 mmol) in methanol (20 ml) was added to methylglyoxal aqueous solution (1) (580 l, 3.8 mmol). The reaction mixture was stirred at room temperature for 2 h. The solvent was then removed under reduced pressure, and the residue was dissolved in methanol. Silica gel was added, and the
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methanol was removed. Silica gel was poured into a glass column, and the bound material was eluted with ethyl acetate:methanol (9:1, v/v). The eluate was monitored by thin layer chromatography (solvent system ethyl acetate/methanol 9:1). The Rf value for the product was 0.1. Fractions with products were pooled, the solvent evaporated, and the mixture was suspended in methanol, absorbed on silica gel and re-chromatographed as before. Fractions with products were combined, dried and stored at 4 °C until use. The puriWed product was found to be an isomeric mixture of 3a and 3b in a 7:3 ratio (»70% yield). The 1H NMR spectrum showed the following characteristics. 3a (major): 1H NMR (DMSO) 8.96 (s, 1H), 8.58 (d, J D 1.8 Hz, 1H), 8.27 (dd, J D 8.8, 2.0 Hz, 1H), 8.09 (d, J D 8.7 Hz, 1H), 2.73 (s, 3H). 3b (minor): 1H NMR (DMSO) 8.96 (s, 1H), 8.52 (d, J D 1.8 Hz, 1H), 8.24 (dd, J D 8.6, 1.8 Hz, 1H), 8.14 (d, J D 8.8 Hz, 1H), 2.73 (s, 3H). The m/z of the product as the trimethylsilyl (TMS) derivative determined by gas chromatography/mass spectrometry was 260, which is compatible with single TMS derivatization on the carboxyl group of 2-methylquinoxaline-6-carboxylate (3a) and 3-methylquinoxaline-6-carboxylate (3b). Conjugation of (3) to keyhole limpet hemocyanin Conjugation was achieved using carbodiimide. Thirty milligrams of (3) was dissolved in 500 l DMSO, and the sample was resuspended in 1.0 ml of 0.1 M MES buVer (pH 6.0). EDC (28.8 mg) was then added in 350 l of water, followed by 2.2 mg of sulfo-NHS in 50 l of water and mixed well. Finally, 20 mg/ml of keyhole limpet hemocyanin (KLH) was added, and the mixture was incubated at room temperature for 4 h. The protein was then extensively dialyzed against PBS. A similar conjugation was made with RNase A to give RNase A-(3). Preparation of monoclonal antibody Mice were injected intraperitoneally with 30 g KLH-(3) in Freund’s complete adjuvant; the initial sensitization was followed with three booster injections of 30 g antigen in incomplete adjuvant at 3 weeks intervals. Serum antibody titers were checked by a direct ELISA. For this determination, 96-well microplate wells were coated with RNase A-(3), and diluted antiserum was incubated with HRP-conjugated rabbit anti-mouse IgG. The mouse with the highest antibody titer was chosen for hybridoma production. Mouse spleen cells were fused with P3U1 myeloma cells by a standard method [26]. Hybridoma supernatants were screened by ELISAs using two diVerent coating solutions. The microplate wells were coated with either 1 g/well RNase A-(3) in 0.05 M sodium carbonate buVer (pH 9.7) (BuVer A) or with 1 g/well RNase A + ribose + OPD in BuVer A. RNase A + ribose + OPD was prepared by incubating 10 mg/ml RNase A + 200 mM ribose + 50 mM OPD + 2 mM EDTA + 2 mM DTPA in 0.1 M phosphate buVer (pH 7.4) at 37 °C for 7 days, followed by dialysis
against PBS for 48 h. After blocking with 5% non-fat dry milk (NFDM), 50 l of hybridoma supernatant was added to wells and incubated for 1.5 h, and then washed 3 times with PBS + 0.05% Tween 20 (PBST). The wells were then incubated for 2 h with 50 l of HRP-conjugated rabbit anti mouse IgG (1:5000 dilution). The wells were washed 3 times with PBST and incubated with 3,3⬘,5,5⬘-tetramethylbenzidine substrate at 37 °C. The reaction was stopped by addition of 50 l of 2 N H2SO4, and the color of the product at 450 nm was measured spectrophotometrically. Hybridomas with the strongest reaction against both RNase A-(3) and (RNase A + ribose + OPD) samples were subcloned by serial dilution until all the subclones were immunoreactive. The medium was collected from large-scale cultures of hybridoma-producing subclones, and the antibody was puriWed on a Protein G–Sepharose column (Amersham Biosciences, Piscataway, NJ). The antibody was identiWed and classiWed as IgG1. Incubation of proteins with carbohydrates and OPD Solutions of BSA or RNase A (10 mg/ml) in 0.1 M sodium phosphate buVer (pH 7.4) were Wltered through a 0.2 m Wlter. Glucose or ribose was added to the protein solution to obtain a Wnal concentration of 500 mM, and the samples were incubated at 45 °C overnight. The following day, OPD (100 mM stock solution prepared by dissolving 10.8 mg in 1.0 ml of DMSO) was added to a Wnal concentration of 100 mM, and the samples were incubated for an additional 4 h at 45 °C. The proteins were dialyzed against PBS for 24 h with one change of buVer after 16 h. They were then stored at 4 °C until use. In another experiment, RNase A was incubated with several carbohydrates in the presence or absence of OPD. A stock solution of RNase A was prepared by dissolving 100 mg in 20 ml of 0.1 M phosphate buVer (pH 7.4). This solution was then Wltered through a 0.2 m Wlter, and 1.0 ml was added to each sample. Duplicate samples included unaltered RNase A controls and samples containing 500 mM of D-glucose, D-fructose, D-ribose, or L-ascorbic acid. All the samples were incubated at 45 °C for 16 h. One set of samples was treated with 100 M OPD stock solution (1.0 mg/1.0 ml in 0.1 M phosphate buVer, pH 7.4) and incubated at 35 °C for 16 h. Sodium borohydride reduction of ribose-modiWed RNase A RNase A 10 mg/ml in 0.1 M sodium phosphate buVer pH 7.4 was Wltered through a 0.2 Wlter. Five hundred millimolar ribose was then added and incubated at 37 °C for 24 h. The unbound sugar was removed by Wltering through a 10 kDa cut oV centrifugal Wlter (Millipore corporation, Bedford, MA). The centrifugation (4000g for 10 min) was repeated three times, each time adding 0.5 ml of incubation buVer to the sample. The pH of the sample was adjusted to 8 using 2 N NaOH. The sample was then divided into two equal portions, to one, 100 mM sodium
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borohydride (NaBH4) was added and both portions were incubated at room temperature for 10 min, followed by incubation on ice for 1 h. One hundred millimolar OPD (dissolved in 25 l DMSO) was then added to each, and incubated for an additional 16 h and dialyzed against PBS for 48 h with change of the buVer after 24 h. Protein concentration was estimated by the Bradford Assay. Samples were tested for reaction with the antibody by a direct ELISA (see below). EVect of N-acetyl-L-arginine RNase A (10 mg/ml) in 0.1 M sodium phosphate buVer, pH 7.4, was Wltered through a 0.2 m Wlter. Five hundred millimolar ribose was then added to the solution and incubated for 24 h at 37 °C. The sample was divided into two parts, to one part 100 mM N-acetyl-L-arginine was added. The other part served as control. The samples were further incubated at 37 °C for 16 h. One hundred millimolar OPD (dissolved in 25 l DMSO) was then added to each and incubated for further 16 h at 37 °C. The samples were dialyzed against PBS for 48 h with change of buVer after 24 h and tested for reaction with the antibody by a direct ELISA (see below). Direct enzyme-linked immunosorbent assay (ELISA) Microplate wells were coated with proteins in 0.05 M carbonate buVer (pH 9.7) at a concentration of 1 g/well, incubated at 4 °C overnight and then washed three times with PBST. Before use in the assay, all wells were blocked for 2 h at room temperature with 300 l of 5% NFDM in PBS and washed three times with PBST. The monoclonal antibody (1:500 diluted in 0.1% BSA/PBS) was applied (50 l/well) to each well and incubated for 1.5 h at 37 °C. The wells were then washed three times with PBST and incubated with 50 l of horseradish peroxidase-conjugated rabbit anti-mouse IgG (Promega, Madison, WI, diluted 1:2500) for 1.5 h at 37 °C. The enzyme reaction was assessed by addition of 100 l of 3,3⬘,5⬘,5⬘-tetramethylbenzidine (Sigma) followed by addition of 50 l of 2 N H2SO4. Chromophore absorbance was measured at 450 nm. Competitive ELISA All procedures were similar to the direct ELISA except that the test samples were incubated with the monoclonal antibody (1:500 in 0.1% BSA/PBS as Wnal dilution) for 1.5 h at 37 °C before addition (50 l) to the wells. Western blotting RNase A samples incubated as described above were subjected to Western blotting (10 g of protein on 12% reducing gels), and the proteins were electrophoretically transferred to a nitrocellulose membrane 0.45 m (Bio-Rad). Identical gels
189
were stained with BioSafe Coomassie blue (Bio-Rad). The membrane was then blocked with 5% NFDM in PBS for 2 h and incubated at 4 °C for 16 h with shaking. The membranes were washed Wve times with PBST and the monoclonal antibody was applied at a dilution of 1:80 in 5% NFDM in PBST. The samples were incubated with shaking for 3 h at room temperature. The membrane was washed again and incubated for 1.5 h longer with rabbit anti-mouse IgG (dilution 1:7500, Promega, Madison, WI). After washing three times, Supersignal West Pico chemiluminescent substrate (Pierce) was applied to the membranes for 10 min, then they were exposed to X-ray Wlm (Pierce, CL-XPosure Film). Isolation of antibody-reactive products from reaction mixture of N-hippuryl-L-lysine + ribose + OPD N-hippuryl-L-lysine (6 mmol) and DTPA (0.013 mmol) were dissolved completely in 2.5 ml of 0.1 M sodium phosphate buVer (pH 7.4). OPD (4 mmol) was dissolved separately in 100 l of DMSO and added to the incubation mixture. D-ribose (2 mmol) was then added to the mixture, and the volume was adjusted to 5.0 ml by addition of 2.5 ml of 0.1 M phosphate buVer (pH 7.4). The mixture was incubated at 70 °C for 48 h. A sample (60 l) of reaction mixture was injected into a C18 semi-preparative reversed phase column (Vydac, 218TP1010). A linear gradient was established with solvent A (0.1% triXuoroacetic acid in water) and solvent B (50% acetonitrile and 0.1% triXuoroacetic acid in water). The following program was applied: 0–5 min, 0% B; 5–60 min, 0–100% B; 60–67 min, 100% B; 68–75 min, 0% B at a Xow rate of 2.0 ml/min. We used an online UV detector (Jasco, Tokyo, Japan, Model UV-970) to monitor the column eZuent for absorbance at 228 nm. Absorbance from quinoxaline-like products was also monitored at 325 nm. Based on the chromatograms, we identiWed six peaks (peaks 2–7) as possible quinoxalines (Fig. 7). They were collected and dried in a Savant Speedvac concentrator (Savant Instruments, Hicksville, NY), re-suspended in 250 l of water and tested by competitive ELISA (only 2–6 were tested, 7 was in insuYcient quantity). 1 H NMR spectroscopy was performed on either Varian Gemini 200 or Varian Inova 400 Instruments (with chemical shifts referenced to TMS or the solvent peak). High-resolution mass spectra (HRMS) were obtained at 20–40 eV on a Kratos MS-25A instrument. Results We planned to detect dideoxyosones in glycated proteins by trapping them with OPD (Fig. 2). This reaction produces quinoxalines. e.g., with glucose derived dideoxyosone, the OPD reaction produces N6-(2,3-dihydroxy-4-quinoxalin-2-ylbutyl)lysine. However, if the Amadori is derived from ribose, the resulting dideoxyosone, upon reaction with OPD would produce N6-(2-hydroxy-3-quinoxalin-2-ylpropyl)lysine and possibly N6-[2-hydroxy-2-(3-methylquinoxalin-2-yl)ethyl]lysine [22,23]. We wanted to raise an antibody
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Fig. 2. Trapping of dideoxyosone intermediates by OPD. Structures in the box were ones we wanted to detect.
that recognized only the quinoxaline moiety, irrespective of substitutions on the quinoxaline core. Accordingly, we synthesized 2-methylquinoxaline-6-carboxylate for conjugation with KLH. The KLH-quinoxaline was then used as an antigen to stimulate production of the monoclonal antibody. To develop an ELISA for screening proteins, we Wrst tested immunoreactivity of BSA and RNase A following incubation with glucose or ribose and OPD. Fig. 3A shows the results of a direct ELISA with 1 g of the protein/well. RNase A + ribose + OPD showed the highest immunoreactivity followed by BSA + ribose + OPD. Proteins modiWed by glucose and OPD were relatively poor reactants. To test if the antibody reacted non-speciWcally with ribose modiWed RNase A, we performed a direct ELISA. The idea was to reduce DDOs with NaBH4 and prevent their reaction with OPD to form quinoxalines. The ELISA procedure involved coating of microplate wells with 1 g of NaBH4 reduced ribose-modiWed RNase A (and treated with OPD) or nonreduced ribose-modiWed RNase A (treated with OPD). The results are shown in Fig. 3B. Antibody failed to react with the reduced protein, but reacted with non-reduced protein. The data suggest that the antibody requires quinoxalines for reaction in glycated proteins. Since the antibody reacted stronger with RNaseA + ribose + OPD (Fig. 3A), we used this preparation in all our subsequent ELISAs. To test the formation of dideoxyosones with various sugars and ascorbate oxidation products, we incubated RNase
Fig. 3. (A) Reactivity of RNase A modiWed by glucose or ribose (+OPD). RNase A was incubated with sugars in the presence of OPD and tested for reaction with antibody by a direct ELISA. (B) Reactivity of NaBH4 reduced RNase A + ribose + OPD. RNase A was incubated with ribose and reduced with NaBH4 before addition of OPD.
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A with glucose, fructose or ascorbate in the presence or absence of OPD. Fig. 4A shows that among these three carbohydrates, fructose produces the greatest quantity of dideoxyosones, followed by glucose and ascorbate. Samples with fructose + OPD (10 g protein/well) inhibited antibody binding by nearly 80%. At this concentration of protein, binding was inhibited 72% by glucose + OPD and 52% by ascorbate + OPD. Samples containing no OPD, RNase A + OPD, or RNase A alone reacted only weakly with the antibody. The mild reaction of the antibody with glycated proteins (without OPD) suggested the possibility of non-speciWc reaction, possibly with AGEs. We tested this possibility by performing a direct ELISA using RNase A + ribose + Nacetyl-L-arginine sample. This sample is expected to contain signiWcant quantities of AGEs, including DDO-derived lysine–arginine crosslinking structures. Our antibody failed to react with this preparation (Fig. 4B). These data suggest that the mild immunoreactivity of glycated proteins (without OPD) may not be due to arginine-derived AGEs. Formation of ‘quinoxaline-like’ products without the involvement of arginine is a possibility. However such products, even if formed, may not signiWcantly interfere with detection of DDO-derived quinoxalines as the reaction with glycated pro-
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teins (without OPD) was 80–90% lower than reaction with glycated proteins (with OPD) (Fig. 4A). We used Western blotting to conWrm the ELISA results. As shown in Fig. 5, all proteins that were modiWed by carbohydrates and OPD showed immunoreactivity. The non-crosslinked RNase A monomer accounted for most of the immunoreactivity, with some in the dimeric protein. These results conWrm our Wndings with ELISA and suggest that OPD curtails crosslinking of proteins through dideoxyosone intermediates. Furthermore, these results indicate that dideoxyosones form not only from sugars, but also from ascorbate, possibly from ascorbate oxidation products. We characterized the antibody further using commercial quinoxalines (0–2000 pmol) in a competitive ELISA. Fig. 6 shows that most quinoxalines reacted with the antibody; the exception was quinoxaline-2-carboxylate. Reaction with 2-methylquinoxaline was strongest, with 100% inhibition of the antibody binding to wells at 500 pmol. Quinoxaline and 5-methylquinoxaline were less eVective than 2-methylquinoxaline and required four times as much (2000 pmol/well) to achieve 100% inhibition of the antibody. Other quinoxalines such as 2-quinoxalinol, 2,3-
Fig. 4. (A) Immunoreactivity of RNase A modiWed by sugars and ascorbate in the presence or absence of OPD. RNase A was incubated Wrst with 500 mM concentrations of sugars or ascorbate for 12 h and then with 1.0 mM OPD at 37 °C and pH 7.4 for 16 h. Proteins were tested in a competitive ELISA using 0, 2, and 4 g protein. (B) EVect of addition of N-acetyl-L-arginine on the immunoreactivity of glycated protein. RNase A was incubated with ribose for 24 h, 100 mM OPD or N-acetyl-L-arginine was then added to the reaction mixture and incubated for 16 h. The reaction mixtures were dialyzed against PBS and tested by direct ELISA as described in Materials and methods.
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Fig. 5. Western blotting of RNase A modiWed by sugars or ascorbate (§OPD). Proteins were incubated under conditions described in Fig. 4. Ten micrograms protein was subjected to SDS–PAGE on a 12% reducing gel and transferred to a nitrocellulose membrane. The membrane was incubated Wrst with the monoclonal antibody followed by incubation with HRP-conjugated rabbit anti-mouse IgG and developed by the Pierce Enhanced Chemiluminescence kit. (A) is a Coomassie stained gel, and (B) is a Western blot of the same material. Lane 1, molecular weight markers; lane 2, RNase A; lane 3, RNase A + OPD; lane 4, RNase A + glucose; lane 5, RNase A + glucose + OPD; lane 6, RNase A + ribose; lane 7, RNase A + ribose + OPD; lane 8, RNase A + fructose; lane 9, RNase A + fructose + OPD; lane 10, RNase A + ascorbate; lane 11, RNase A + ascorbate + OPD.
Fig. 6. Immunoreactivity of quinoxalines with the antibody. Commercially available quinoxalines (0–2000 pmol) were tested in competitive ELISA.
dimethylquinoxaline, and quinoline were comparable, producing 80–90% inhibition at 2000 pmol/well. From these results, we concluded that the antibody favors 2-methyl substitution on quinoxaline for maximum reactivity (Fig. 6, inset).
To determine the possible epitope structures in lysine-derived dideoxyosones, we attempted to isolate antibody-reactive structures following the incubation of Nhippuryl-L-lysine with ribose and OPD. The reaction mixture was subjected to HPLC on a C18 reversed phase column. The column eluate was monitored for absorbance at 228 nm (for the hippuryl group) or 325 nm (for quinoxalines). Several peaks were observed as shown in Fig. 7. Peaks 2–7 were observed at both 228 and 325 nm, indicating that they could be quinoxalines. These peaks were collected from 2 injections and dried in a Savant Speed Vac Concentrator. The dried fractions were re-suspended in 0.25 ml of dH2O. From each fraction, 100 l of sample was mixed with 100 l of 6 N HCl and incubated at 110 °C for 16 h. After acid hydrolysis, drying of samples and reconstitution in 100 l of dH2O, 20 l from each was spotted on a silica gel thin layer chromatography plate. The unhydrolyzed portion of the fractions was spotted on a separate plate and developed by spraying ninhydrin reagent (2% in ethyl alcohol) onto each plate. Fractions 4, 5, and 6 were weakly ninhydrin positive after acid hydrolysis, indicating that these peaks contain lysine. Products in peaks 2 and 3 were ninhydrin negative and thus are probably lysine-free quinoxalines, formed from the reaction of deoxyosone intermediates of glycation with OPD. We tested products in peaks 4–6 in a competitive ELISA to determine if they reacted with the antibody. The results show that the product in peak 5 was most reactive with nearly 75% inhibition of antibody binding at 0.4 l (Fig. 8). Products in peak 4 and 6 also inhibited antibody binding, but were less eVective than the product in peak 5. We acknowledge that these measurements are not quantitative, because product concentrations presumably diVer within the peaks. Based on these results, we collected products in peaks 4, 5, and 6 (henceforth referred to as compounds 4, 5, and 6) from 30 injections of 60 l each. These samples were concentrated by lyophilization and re-injected into the HPLC column. The separated compounds were then collected, freeze-dried and dissolved in 0.5 ml methanol. The 1H NMR spectrum for 4 and 5 showed the following signals: 4: 1H NMR (CD3OD) 9.07 (s, H-13), 8.15– 8.05 (2H, H-14,17), 7.90–7.80 (4H, H-3,15,16), 7.55–7.40 (3H, H-1,2), 4.50–4.30 (2H, H-5,11), 4.10–4.00 (m, 2H, H-
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Fig. 7. Isolation and puriWcation of immunoreactive products from reaction mixture of N-hippuryl-L-lysine + ribose + OPD by HPLC. HPLC was performed on a C18 reversed phase semi-preparative column using a water/acetonitrile (with 0.1% TFA) gradient program. The column eluate was monitored for absorbance at 228 nm (for the hippuryl group, A) and 325 nm (for quinoxalines, B). Peaks 4–7 were collected by repeated injections. The products in these peaks were further puriWed on the same column as described in Materials and methods.
Fig. 8. Immunoreactivity of HPLC puriWed products. The products puriWed from HPLC (peaks 4–6) were freeze-dried and dissolved in DMSO. Samples at indicated volumes were tested for reaction with the antibody in a competitive ELISA.
4), 3.20–2.90 (6H, H-9,10,12), 2.00–1.40 (6H, H-6,7,8); 5: 1 H NMR (CD3OD) 8.80 (s, H-13), 8.20–8.00 (2H, H14,17), 7.90–7.70 (4H, H-3,15,16), 7.60–7.40 (m, 3H, H-1,2),
4.60–4.45 (2H, H-5,11), 4.10–3.90 (m, 2H, H-4), 3.20–2.90 (6H, H-9,10,12), 2.00–1.40 (6H, H-6,7,8). Fig. 9A shows the 1H NMR spectrum for 5, the one for 4 is nearly identical (not shown). Compounds 4 and 5 are diastereoisomers caused by two chiral centers, C-5 and C-11. The proton signals of 4 and 5 around 9.00 ppm were assigned to H-13 due to the strong deshielding eVect of the quinoxaline ring. The resonances around 7.5–8.2 ppm region are due to the hippuryl and quinoxaline moieties. The high Weld aliphatic hydrogen signals between 2.00 and 1.40 ppm are attributed to the lysine side chain internal methylenes. HRMS (FAB) for 5 showed a m/z of 494.2390, which was similar to the calculated m/z for C26H32N5O5 (MH+) of 494.2403. The UV spectrum of 5 showed max of 325 nm, which was attributable to the quinoxaline ring (Fig. 9B). The UV spectrum for 4 was identical to that of 5. Based on these characteristics, the product was identiWed as, N1benzoylglycyl-N6-(2-hydroxy-3-quinoxalin-2-ylpropyl)lysine (structure shown in Fig. 9A inset). The 1H NMR analysis of 6 showed the following signals (Fig. 10A): 1H NMR (CD3OD) 8.20–7.90 (2H, H14,17), 7.90–7.70 (4H, H-3,15,16), 7.60–7.30 (3H, H-1,2), 5.40–5.30 (m, 1H, H-11), 4.70–4.50 (m, 1H, H-5), 4.20–3.90
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Fig. 9. 1H NMR spectrum of 5. Compound 4 displayed a nearly identical spectrum to 5. Based on the 1H NMR characteristics, the structure of the product is shown in the inset. UV spectrum is shown in the (B).
(m, 2H, H-4), 3.80–3.10 (4H, H-9,10), 2.90 (s, 3H, H-12), 2.10–1.50 (6H, H-6,7,8). The structure of 6 was determined by the speciWc positions of H-11 and H-12 besides its aromatic protons attributed to the hippuryl and quinoxaline groups. Due to the deshielding eVect of quinoxaline ring and electronegativity of hydroxyl group, H-11 resonates downWeld around 5.3 ppm. The 12-CH3 singlet appears at 2.9 ppm, characteristic of the methyl group at position 2 on the quinoxaline ring. HRMS (FAB) showed a m/z of 494.2406, which was fully agreeable with the calculated m/ z for C26H32N5O5 (MH+) of 494.2403. The UV spectrum of 6 showed max of 325 nm, which was due to the quinoxaline ring (Fig. 10B). Based on these characteristics, we identiWed the product as N1-benzoylglycyl-N6-[2-hydroxy2-(3-methylquinoxalin-2-yl)ethyl]lysine (structure shown in Fig. 10A inset). Fig. 11 indicates the potential mechanism of formation of quinoxalines from the reaction of N-hippuryl-L-lysine + ribose + OPD. Discussion Dideoxyosones are likely to be major intermediates during AGE synthesis in vivo, yet little is known about the chemical pathways of AGE formation from these intermediates. Only two dideoxyosone-derived AGEs, pentosidine
and glucosepane, are hitherto known; they are both crosslinking structures. Biemel and Lederer [25] suggested that glucosepane accounted for only a fraction of the total dideoxyosone formed from the reaction of glucose, and they speculated that dideoxyosone could generate a variety of other protein crosslinks. In this regard, detection of dideoxyosones is an important step towards understanding how this pathway contributes to the pathogenesis of diabetic and age-associated complications. Biemel and Lederer [25] showed that OPD was better than aminoguanidine in trapping dideoxyosones. Accordingly, we chose OPD to trap dideoxyosones. We reasoned that some quinoxalines might have 2-methyl group substitution as shown in Fig. 2. We wanted to detect dideoxyosones (as quinoxalines) formed from not just sugars, but also from ascorbate oxidation products. Taking these factors into consideration, we used 2-methylquinoxaline-6carboxylate as an antigen. The antigen was coupled to KLH through the COOH group. We found that this monoclonal antibody recognized a variety of quinoxalines, but the favored structure was 2-methylquinoxaline. We assumed that when dideoxyosones formed on proteins react with OPD, they would generate quinoxalines with the sugar moiety attached to the pyrazine ring. However, our antigen, 2-methylquinoxaline-6-carboxylate was
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Fig. 10. 1H NMR spectrum of 6. UV spectrum is shown in (B).
coupled to protein through the carboxyl group on the benzene rather than pyrazine ring. This diVerence may preclude or weaken antigen detection in biological specimens. However, the fact that we could detect quinoxalines in proteins incubated with sugars (+OPD) suggests that the antibody recognizes ‘biological’ quinoxalines, and it might prove useful for assessing the impact of dideoxyosones in AGE formation in vivo. Formation of greater quantities of dideoxyosones from fructose than from glucose may reXect the higher reactivity of fructose with lysine. Fructose-mediated glycation is reported to produce at least 10 times greater crosslinking than glucose-mediated glycation [27]. The formation of dideoxyosones from ascorbate is not totally unexpected, because ascorbate through oxidation forms sugars, such as threose [28], which can potentially form dideoxyosones, like ribose and glucose. Our Western blot experiment showed that the antibody recognizes primarily the modiWed monomer of RNase A. Coomassie staining conWrmed that a larger portion of the protein remained as a monomer after reaction with sugars and OPD relative to reaction of sugars in the absence of OPD. This suggests that signiWcant pro-
tein crosslinking occurs through dideoxyosone intermediate formation and that OPD inhibits protein crosslinking by binding to dideoxyosones. The identiWcation of N1-benzoylglycyl-N6-(2-hydroxy-3quinoxalin-2-ylpropyl)lysine and N1-benzoylglycyl-N6-[2hydroxy-3-(3-methylquinoxalin-2-yl)ethyl]lysine as major antigens from the reaction of N-hippuryl lysine + ribose + OPD conWrms that the precursor dideoxyosone lysine adducts, capable of crosslinking to protein arginine residues, form from ribose during glycation. Whether these are the major products generated during AGE formation in vivo remains to be established. We believe that quinoxaline-antibody detection of dideoxyosone intermediates oVers considerable advantages over conventional chromatographic methods. The antibody can recognize quinoxalines of dideoxyosones derived from a variety of sugars, and it can be used to quantify dideoxyosones derived from various Maillard reaction pathways. The ability of our monoclonal antibody to detect dideoxyosone intermediates as quinoxaline derivatives oVers a rapid screening method for future research as well as the potential for therapeutic agents that could be used against AGE
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Fig. 11. Possible pathways for the formation of quinoxalines (4, 5, and 6) from the reaction of N-hippuryl-L-lysine, ribose, and OPD.
formation from dideoxyosones intermediates. Such agents would be an important addition to the therapeutic armamentarium against diabetes. Acknowledgments This study was supported by NIH Grants R21DK068045, R01EY-09912, and R01EY-016219 (R.H.N.), P30EY-11373 (Visual Sciences Research Center of CWRU), R01-AG14249 (L.M.S.), Research to Prevent Blindness, NY and Ohio Lions Eye Research Foundation. References [1] S.R. Thorpe, J.W. Baynes, Amino Acids 25 (2003) 275–281. [2] M. Brownlee, Metabolism 49 (2000) 9–13. [3] A.W. Stitt, A.J. Jenkins, M.E. Cooper, Expert Opin. Invest. Drugs 11 (2002) 1205–1223. [4] M.C. Wells-Knecht, S.R. Thorpe, J.W. Baynes, Biochemistry 34 (1995) 15134–15141. [5] H. Vlassara, M.R. Palace, J. Intern. Med. 251 (2002) 87–101. [6] E.B. Frye, T.P. Degenhardt, S.R. Thorpe, J.W. Baynes, J. Biol. Chem. 273 (1998) 18714–18719. [7] J.A. Dunn, D.R. McCance, S.R. Thorpe, T.J. Lyons, J.W. Baynes, Biochemistry 30 (1991) 1205–1210. [8] D.G. Dyer, J.A. Dunn, S.R. Thorpe, K.E. Bailie, T.J. Lyons, D.R. McCance, J.W. Baynes, J. Clin. Invest. 91 (1993) 2463–2469. [9] F.A. Shamsi, A. Partal, C. Sady, M.A. Glomb, R.H. Nagaraj, J. Biol. Chem. 273 (1998) 6928–6936. [10] R.H. Nagaraj, D.R. Sell, M. Prabhakaram, B.J. Ortwerth, V.M. Monnier, Proc. Natl. Acad. Sci. USA 88 (1991) 10257–10261. [11] P. Chellan, R.H. Nagaraj, J. Biol. Chem. 276 (2001) 3895–3903. [12] O.K. Argirov, B. Lin, B.J. Ortwerth, J. Biol. Chem. 279 (2004) 6487–6495.
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